Zonulin and blood–brain barrier permeability are dissociated in humans

Dear Editor, Zonulin, or prehaptoglobin-2, mediates intestinal permeability in coeliac disease through the regulation of epithelial tight junctions.1 Tight junction breakdown at the blood–brain barrier (BBB) is a common pathological finding in neurological disease,2 and several in vitro and preclinical in vivo studies have suggested that zonulin plays a role inmodulation of BBBpermeability,3–6 yet using multiple methods, we here consistently find that zonulin plays a negligible role in human BBB permeability. Zonulin is a member of the MASP (mannose-binding lectin-associated serine protease) family of proteins, and elevated serum zonulin levels have been reported in a number of neurological conditions such as multiple sclerosis7 and Alzheimer’s disease.8 The significance of zonulin upregulation in these neurological diseases is not certain. Specifically, it is not clear whether zonulin is an epiphenomenon, or has an effect on the brain, whether directly or mediated through the gut–brain axis. To study the association between zonulin and BBB permeability in healthy individuals and patients with neurological disease, we employed two techniques to measure permeability across a range of molecular weights: QAlb, or the quotient of cerebrospinal fluid to serum albumin (60 000 Da) in Study A and dynamic contrastenhanced magnetic resonance imaging with gadobutrol tracer (600 Da) in Study B (Supplemental Methods). Participant characteristics are shown in Table 1. Out of a total of 217 cases (including people with neurological conditions and control individuals) across both studies, 58 (27%) individuals tested negative for serum zonulin using a novel enzyme-linked immunosorbent assay (ELISA) (Supplemental Methods). Serum zonulin concentration followed a non-Gaussian distributionwith a range of 0–11 μg/ml. Therewere no effects of sex, age or disease status (disease vs. control) on zonulin concentrations (analysis of covariance, F(3,216) = 1.06, p = .37). In order to assess zonulin using a complementary dual approach,

we determined the haptoglobin phenotype (Supplemental Methods) across all participants from both Studies A and B. As zonulin is a precursor of haptoglobin-2, one would expect an increase in serum zonulin concentration with HP2 allele dosage (HP1-1 < HP2-1 < HP2-2), and this pattern was indeed observed (Figure S1), with a significant difference in zonulin among haptoglobin phenotypes (analysis of variance, F(2,216) = 43.8, p < .0001). However, it is important to note that 9 out of 34 HP1-1 cases tested positive for zonulin ( Figure S1), indicating that the ELISA was cross-reacting against other ZFP (zonulin family of proteins) members, as established previously with other zonulin ELISAs. 9 This highlights the importance of adopting a dual approach, when assessing zonulin, by using both ELISA and haptoglobin phenotyping.
In Study A, Q Alb was significantly higher with age (p = .0002), higher in males versus females (p < .0001) and higher in people with neurological disease versus healthy controls in univariable analysis (p = .0003, Figure 1A-C). A multivariable linear regression, controlling for age, sex and disease status, showed that serum zonulin did not associate with Q Alb (p = .92, Table 2 and Figure 1D). Regressing Q Alb on the presence or absence of the HP2 allele (HP2-2 and HP2-1 individuals vs. HP1-1 individuals) instead of zonulin concentration, using the same covariates, also showed no association (p = .313, data not shown).
In Study B, K i was significantly higher with age (p = .007), was not different between males versus females (p = .23) and was significantly higher in people with multiple sclerosis versus healthy individuals in univariable analysis (p = .001, Figure 1E-G). A multivariable linear regression, controlling for sex, disease status and age, showed that serum zonulin was not positively associated with K i (Table 2 and Figure 1H). Regressing K i on the presence or absence of the HP2 allele (HP2-2 and HP2-1 individuals vs. HP1-1 individuals) instead of zonulin concentration also showed no association (p = .50, data not shown). Most circulating mediators relevant to .06 (.05) Note: Age is given as the mean, zonulin, Q Alb and K i are given as medians (interquartile range). a Diagnoses for participants with neurological disease in Study A included: inflammatory disease (n = 79), degenerative disease (n = 13), ischaemic disease (n = 13), normal pressure hydrocephalus (n = 9), infectious (n = 5), headache syndrome (n = 5), tumour (n = 2), structural (n = 2), epilepsy (n = 1), idiopathic (n = 1), hereditary neuropathy (n = 1), metabolic (n = 1), vascular (n = 1) and unknown (n = 21). The effect of zonulin on the permeability of the hCMEC/D3 monolayer to 70 and 150 kDa fluorescent F I G U R E 1 Study A employed Q Alb as a human blood-brain barrier (BBB) permeability marker. Q Alb was higher with age (A) and in males (B) and individuals with neurological disease (C), in univariable analyses. (D) Multivariable linear regression showed no relationship between zonulin and Q Alb . Study B used dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to derive K i as a measure of human BBB permeability. K i was significantly higher with age (E), was not different between males and females (F) and was significantly higher in individuals with multiple sclerosis versus healthy individuals (G) in univariable analyses. (H) Multivariable linear regression showed no relationship between zonulin and K i . As K i in healthy individuals is close to zero, negative values may arise due to random noise. No positivity constraint was applied to the data. In (A), (D), (E) and (H), dashed lines represent 95% confidence intervals.

TA B L E 2 Multivariable linear regression results
dextrans was assessed at 1-h intervals up to 6 h after treatment with recombinant zonulin. A 1:1 mixture of the cytokines TNF-α (tumour necrosis factor-alpha) and IFN-γ (interferon-gamma) was used as a positive control. Com-pared to vehicle control wells, TNF-α and IFN-γ significantly increased the permeability of the hCMEC/D3 monolayer to the 70-kDa dextrans (Figure 2A,C) and 150-kDa dextrans ( Figure 2B,D). Monolayers treated with zonulin showed no difference in permeability to the 70-kDa dextrans (Figure 2A,C) and 150-kDa dextrans ( Figure 2B,D) compared with controls. This is the first study to examine the role of zonulin in BBB permeability in humans. A major strength of this work, important in confirming the absence of a significant contribution of zonulin to BBB regulation, is the robustness of findings using different methodologies. Still, it remains possible that local and/or transient changes in concentrations of zonulin at brain capillary surfaces are not well represented by either circulating zonulin levels or haptoglobin phenotype. The simplistic in vitro model of the BBB used does not fully recapitulate the anatomy and physiology of the living BBB, and future studies should aim to replicate results using three-dimensional all-human multicellular BBB models and more sophisticated methods for assessing BBB permeability.
Although preclinical studies suggested that zonulin has potential to regulate BBB permeability, 3-6 we find no evidence for a significant contribution of zonulin in humans, using a number of technical approaches to account for zonulin and to quantify BBB permeability. This is an important negative finding and suggests that the association of serum zonulin levels with clinical manifestations in various neurological diseases 7,8 is unlikely to be mediated by a direct effect of zonulin on BBB permeability. Other indirect mechanistic pathways such as gastrointestinal permeability linked with the gut-brain axis are more likely to be responsible, as exemplified by zonulin transgenic mice which display neurological abnormalities improved by antibiotic depletion of gut microbiota. 10 Future studies should further investigate these pathways and the relationship between zonulin and the severity of neurological disease.

A C K N O W L E D G E M E N T S
Dr Craig Sturgeon for recombinant zonulin synthesis, Bio-Rad (Montpellier, France) for supplying the zonulin ELISA, Prof Nacho Romero and Dr Eduardo Frias for advising on the transwell assay, Dr Patrick Garland for sharing the haptoglobin phenotyping protocol, Angela Dareka and Maria Liljeroth for facilitating magnetic resonance imaging in Study B and Monica Fenn, Elisabeth Jarman, Raeisa Ali, Chloe Sayce, Ellen Adams, Connie Temple-Brown and Aimee O'Neill for patient recruitment to Study A.

C O N F L I C T O F I N T E R E S T
None of the authors have any potential competing interests.


Insights into the Mechanisms of Action of Proanthocyanidins and Anthocyanins in the Treatment of Nicotine-Induced Non-Small Cell Lung Cancer

In traditional medicine, different parts of plants, including fruits, have been used for their anti-inflammatory and anti-oxidative properties. Plant-based foods, such as fruits, seeds and vegetables, are used for therapeutic purposes due to the presence of flavonoid compounds. Proanthocyanidins (PCs) and anthocyanins (ACNs) are the major distributed flavonoid pigments in plants, which have therapeutic potential against certain chronic diseases. PCs and ACNs derived from plant-based foods and/or medicinal plants at different nontoxic concentrations have shown anti-non-small cell lung cancer (NSCLC) activity in vitro/in vivo models through inhibiting proliferation, invasion/migration, metastasis and angiogenesis and by activating apoptosis/autophagy-related mechanisms. However, the potential mechanisms by which these compounds exert efficacy against nicotine-induced NSCLC are not fully understood. Thus, this review aims to gain insights into the mechanisms of action and therapeutic potential of PCs and ACNs in nicotine-induced NSCLC.

Introduction
Lung cancer (LC) is considered the main diagnosed cancer causing death worldwide [1]. LC is broadly categorized into two major histologic classes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC is further subclassified into squamous, adenocarcinoma and large cell carcinoma, which constitute 85% of smokingattributable LC cases [2,3]. Tobacco products contain several carcinogenic compounds, such as tobacco-specific nitrosamines (i.e., NNN and NNK), which enhance production of DNA adducts in the lungs of smokers, thereby causing mutations of several NSCLC suppressor genes including protein p53 [4][5][6]. Although nicotine is considered non-carcinogenic, it may contribute to NSCLC development [7][8][9][10][11]. Nicotine enhances proliferation, angiogenesis and metastasis and inhibits apoptosis/autophagy in NSCLC cells by activating nicotinic acetylcholine receptors (nAChRs), especially the α7 subunit, and its downstream signaling pathways including the proto-oncogene serine/threonine kinase (Rb-RAF1), the phosphatidylinositol-3 kinase/serine/threonine kinase (PI3K/Akt), the mammalian target of rapamycin (mTOR), the nonreceptor tyrosine/kinase focal adhesion/protein kinase (Src/FAK/PKC) and the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) [12]. Nicotine also induces epithelial-to-mesenchymal transition (EMT), which is the key step in enhancing tumor progression in NSCLC cells, resulting in upregulation of several transcription and growth factors via activation of α7nAChRmediated signaling pathways [12]. The mechanisms by which nicotine enhances tumor progression in NSCLC cells have been previously described in greater details [13]. In brief, nicotine binds to α7nAChR, activating the cellular signaling pathways involved in proliferation, metastasis, angiogenesis and anti-apoptosis/autophagy, which increases expression of EMT-associated molecules in NSCLC cells such as hypoxia inducible factor-1 (HIF-1α), vascular endothelial growth factor (VEGF), transforming growth factor β (TGF-β), pathways involved in proliferation, metastasis, angiogenesis and anti-apoptosis/autophagy, which increases expression of EMT-associated molecules in NSCLC cells such as hypoxia inducible factor-1 (HIF-1α), vascular endothelial growth factor (VEGF), transforming growth factor β (TGF-β), deca-pentaplegic homolog (Smad), B-cell lymphoma-2 (Bcl-2), metalloproteinases (MMPs), cyclinD1, Snail, twist, vimentin, fibronectin and N-cadherin [13]. Figure 1 summarizes the mechanisms for nicotine in the development of NSCLC. There is no clear recommendations on safe dietary supplements for use in treating NSCLC, particularly in smokers [14]. However, combination therapy of monoclonal antibody-based immunotherapy and chemotherapeutic agents has been shown to be a promising treatment strategy for NSCLC [15]. In addition, natural flavonoids present in fruits (e.g., citrus) in combination with chemotherapeutic agents, such as cisplatin, have shown anti-NSCLC effects, demonstrated by inhibition of the α7nAChR-mediated signaling pathways involved in cellular processes including proliferation, inflammation and anti-apoptosis [13,16].The health benefits of fruits are due to the high levels of bioactive flavonoids they contain, such as PCs and ACNs [17,18]. A large number of studies have documented the health benefits of natural flavonoids derived from fruits and plant foods, in relation to their biological attributes such as anti-diabetes/anti-cancer activity, reducing cardiovascular diseases and improving the blood lipid profile [17][18][19]. Thus, there is a real need to investigate natural flavonoid compounds, such as PCs and ACNs, as therapeutic agents for nicotine-induced NSCLC.
Flavanols include a group of natural compounds categorized according to their chemical structures into catechins and PCs, which are found in various plant-based foods. PCs, also known as condensed tannins, are polymeric and/or oligomeric pigments found in common plant-based foods (e.g., fruits, vegetables, cereal grains, and legumes), Cinnamomi Cortex (barks of Cinnamomum cassia used as a traditional Chinese medicine) and Vaccinium berries, for which a range of therapeutic effects have been reported including anti-cancer, antimicrobial, anti-diabetic, anti-obesity, cardioprotective and antioxidant properties [20]. Procyanidins are the most homo-oligomeric PC derivatives comprised of epicatechin/catechin monomeric, connected via the C4→C6/C4→C8 bond (B-type linkage) and the C2→O7 bond (A-type linkage), and dominated by dimers (e.g., procyanidin A1-A2/B1-B8), trimers (e.g., selligueain A/B and procyanidin C1/C2), and tetramers (degree of polymerization ranged from 5 to 11) [20][21][22].
ACNs, featuring six common glycosylated forms of anthocyanidins (i.e., malvidin, pelargonidin, cyanidin, petunidin, delphinidin and peonidin) with hydroxyl (OH) moieties in their structure at the 3 position on the C-ring are water-soluble pigments belonging to flavonoids responsible for producing various colors in fruits, berries and vegetables There is no clear recommendations on safe dietary supplements for use in treating NSCLC, particularly in smokers [14]. However, combination therapy of monoclonal antibody-based immunotherapy and chemotherapeutic agents has been shown to be a promising treatment strategy for NSCLC [15]. In addition, natural flavonoids present in fruits (e.g., citrus) in combination with chemotherapeutic agents, such as cisplatin, have shown anti-NSCLC effects, demonstrated by inhibition of the α7nAChR-mediated signaling pathways involved in cellular processes including proliferation, inflammation and anti-apoptosis [13,16].The health benefits of fruits are due to the high levels of bioactive flavonoids they contain, such as PCs and ACNs [17,18]. A large number of studies have documented the health benefits of natural flavonoids derived from fruits and plant foods, in relation to their biological attributes such as anti-diabetes/anti-cancer activity, reducing cardiovascular diseases and improving the blood lipid profile [17][18][19]. Thus, there is a real need to investigate natural flavonoid compounds, such as PCs and ACNs, as therapeutic agents for nicotine-induced NSCLC.
Flavanols include a group of natural compounds categorized according to their chemical structures into catechins and PCs, which are found in various plant-based foods. PCs, also known as condensed tannins, are polymeric and/or oligomeric pigments found in common plant-based foods (e.g., fruits, vegetables, cereal grains, and legumes), Cinnamomi Cortex (barks of Cinnamomum cassia used as a traditional Chinese medicine) and Vaccinium berries, for which a range of therapeutic effects have been reported including anti-cancer, antimicrobial, anti-diabetic, anti-obesity, cardioprotective and antioxidant properties [20]. Procyanidins are the most homo-oligomeric PC derivatives comprised of epicatechin/catechin monomeric, connected via the C4→C6/C4→C8 bond (B-type linkage) and the C2→O7 bond (A-type linkage), and dominated by dimers (e.g., procyanidin A1-A2/B1-B8), trimers (e.g., selligueain A/B and procyanidin C1/C2), and tetramers (degree of polymerization ranged from 5 to 11) [20][21][22].
ACNs, featuring six common glycosylated forms of anthocyanidins (i.e., malvidin, pelargonidin, cyanidin, petunidin, delphinidin and peonidin) with hydroxyl (OH) moieties in their structure at the 3 position on the C-ring are water-soluble pigments belonging to flavonoids responsible for producing various colors in fruits, berries and vegetables that exert a protective effect against diabetes, cardiovascular and neurodegenerative diseases and cancers (including LC) [23][24][25].
In various plant species, PCs and ACNs share the first and last steps of flavonoid biosynthesis and its regulatory genes including dihydroflavonol reductase, flavanone 3βhydroxylase and chalcone isomerase/synthase [26,27]. PCs and ACNs haveshown to be the key compounds contributing to the intense color of cereals, fruits, vegetables and blueberries [28], and they have been reported to reduce the production of reactive oxygen species (ROS) in some berry fruits, which may make them potential anti-antioxidant agents [29]. Development and ripening of plant species involve complex processes that result in substantial structural and metabolic changes such as changes in texture and flavor formation and pigment accumulation [30]. These processes are coordinated by abscisic acid (ABA) and other ripening-related genes, which regulate PCs and ACNs biosynthesis [30]. Ripening is considered an important stage where major flavonoids are accumulated [31]. A diverse range of plant phytohormones including cytokinin, gibberellins, auxin, jasmonic acid and brassinosteroids are involved in regulating different aspects of fruit development and ripening through multilevel regulatory mechanisms [31]. PCs are the main compounds detected at the early stage of plant development, whereas ACNs accumulate early in ripening [31]. ACNs act as the major colored pigments attracting animals and insects for seed dispersal in berry fruits [30]. In climacteric fruit ripening, ethylene plays a key hormonal role in stimulating the differential expression of many gene encoding transcription factors that regulate pigments accumulation, cell wall softening, texture change and aroma, flavor and skin color development [32,33].
A recent reviews showed that particular flavonoid compounds (e.g., quercetin, kaempferol, epigallocatechin gallate, and delphinidin) derived from plant-based foods act against NSCLCin in vitro/in vivo models [16,34]. However, the molecular mechanism of PCs and ACNs with regard to their preventive effects against nicotine-induced NSCLC have not been fully elucidated. A recent review showed that flavone-rich extracts from Scutellaria baicalensis, and baicalein, baicalin, wogonin, wogonoside and oroxylin A, in particular, serve as potential adjuvant agents for the treatment of nicotine-induced NSCLC. These compounds exert anti-inflammatory, anti-angiogenesis, anti-proliferative and apoptotic/autophagic effects through inhibiting several cellular signaling pathways as potential molecular mechanisms [13].
The therapeutic effects of PCs and ACNs against nicotine-induced NSCLC may depend on their bioavailability, that is, absorption, distribution and excretion. ACNs are distinguishable from other flavonoids because of its apparent low bioavailability [35,36]. However, studies have demonstrated that ACNs have higher bioavailability and bioactivity in vitro than in vivo [37]. ACNs are absorbed in the blood, distributed to target tissues and excreted into bile as the glycosylated structure [35]. The absorption efficiencies of the hydrophobic ACNs are greater than the hydrophilic ones in vitro [38]. In vitro studies showed that PAs oligomers are degraded into monomers and dimers in acidic gastric juice by human fecal microflora, which are then absorbed as intact molecules in the colon and excreted into urine. In vivo, epicatechin (EC) and methylated EC, as the major metabolites of PAs dimers, are absorbed in the intestinal mucosa in very low contents, catabolized by fecal microflora fermentation into low molecular phenolic acids and then excreted into urine [39]. Given that PCs and ACNs are used as therapeutic agents against many chronic diseases, targeting these compounds might help our understanding of the mechanisms involved in nicotine-induced NSCLC treatment, particularly in smokers. To date, no reviews have discussed the molecular mechanisms of PCs and ACNs in nicotine-induced NSCLC treatment. Thus, this paper highlights the mechanisms of action and therapeutic potential of PCs and ACNs in nicotine-induced NSCLC.

Methods
A literature review was undertaken to identify the published studies exploring the potential molecular mechanisms of PCs and ACNs in nicotine-induced NSCLC treatment from 2000 up to June 2022 via searches of PubMed/MEDLINE. The following search terms were used:"proanthocyanidins" OR "procyanidin" AND "anthocyanins" OR "malvidin" OR "pelargonidin" OR "cyanidin" OR "petunidin" OR "delphinidin" OR "peonidin" AND "NSCLC"OR "lung cancer" AND "nicotine" OR "cigarette smoke". The search included all types of study design published in English. All studies were identified and reviewed by the author. From a total of 202 articles searched using the databases, 30 studies were selected based on the search terms.

Proanthocyanidins in Nicotine-Induced NSCLC Treatment
Several studies demonstrated the therapeutic effects of PC-rich extracts from plantbased foods and/or medicinal plants at different nontoxic concentration against nicotineinduced NSCLC. Cranberry-derived PCs suppress tumor cell growth in NSCLC cells [40,41], but the mechanisms for this action have not been well investigated [40]. Treatment with cranberry PCs resulted in a significant induction of apoptosis and cell cycle arrest in NSCLC cells via upregulating the expression of pro-apoptotic-related markers (e.g., cytochrome c and caspase 3) [42].
PCs from grape seed extract have shown promising results in nicotine-induced NSCLC treatment. For example, using the in vivo proteolysis/antitumor assay and the in vitro proteolysis/angiogenesis assay, PCs inhibit angiogenesis-mediated tumor growth in NSCLC cells, in part by suppressing vascular extracellular matrix (ECM) proteolysis byMMP-2 [43]. Treatment of NSCLC cells with PCs using the in vivo tumor xenograft assay and the in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell proliferation/survival resulted in suppression of cell proliferation in vitro/vivo, and inhibition of angiogenesis and induction the apoptotic cell death of tumor cells in vivo. Such effects are mediated by upregulation of insulin-like growth factor binding protein-3 (IGFBP-3) levels and inhibition of proliferating cell nuclear antigen (PCNA) in the tumor microenvironment [44]. A study used the in vivo tumor xenograft assay and the in vitro cell deathenzyme-linked immunosorbent assay (ELISA) and MTT assay for assessing proliferation of NSCLC cells showed that PCs cause proliferation inhibition and apoptosis induction via the inhibition of cyclooxygenase-2 (COX-2) expression and prostaglandin-2 (PGI-2) receptors in NSCLC cells [45]. The mechanism underlying the anti-migration effect of PCs on NSCLC cells involve inhibiting nitric oxide (NO) synthase, N(G)-nitro-Larginine methyl ester (L-NAME), and the ERK1/2 and MAPK signaling pathways [46]. The anti-proliferative/apoptotic effects of PCs onNSCLC cells are mediated via the activation of caspase 3 expression, prostacyclin synthase (PTGIS)/PGI2 (as measured by 6-keto PGF1α), and 15-lipoxigenase-2/15(S)-hydroxyeicosatetraenoic acid (15-LOX-2)/15-HETE production [47]. PCs showed the inhibitory effects on the cigarette smoke condensate (CSC)-induced migration of NSCLC cells through inhibition of NADPH oxidase (NOX)induced oxidative stress and EMT transition [48]. Treatment with PCs using the colorimetric caspase-3 activity assay in vivo and in vitro showed apoptotic effects through increased expression of pro-apoptotic markers (e.g., poly ADP ribose polymerase (PARP); Bcl-2associated X protein (Bax)), and decreased expression of apoptotic markers (e.g., Bcl-2 and cyclins) [49]. A study used the in vivo tumor xenograft assay and the in vitro MTT and miR-106b ISH assays showed that PCs promote anti-proliferative/invasive effects on NSCLC cells via downregulating miR-106b expression and upregulating cyclin-dependent kinase inhibitor 1A (CDKN1A) mRNA and p21 expression [50].
A few studies on NSCLC cells after treatment with the Cinnamomi Cortex extract PCs showed a significant reduction in nuclear factor-E2-related factor 2(Nrf2) expression, and insulin-like growth factor-1 receptors (IGF-1R) were responsible for induced proliferation [51,52]. Cinnamomi Cortex extract procyanidin C1 exert anti-metastatic activity by suppressing TGF-β-induced EMT in NSCLC cells [53].
Treatment with PCs inhibits hydrogen peroxide (H 2 O 2 )-induced NSCLC cell viability, as shown by reduced ROS and malondialdehyde (MDA) production, hydrogen peroxide(H 2 O 2 )-induced oxidative stress, and promoted the expression of Nrf2 target genes [54]. PCs inhibit proliferation, viability, along with induction of apoptosis and G2/M cell cycle arrest in NSCLC cells. This is triggered by inhibiting the EMT-related molecules (e.g., N-cadherin and vimentin), expression of apoptotic markers (e.g., Bcl-2), and increasing expression of pro-apoptotic markers (e.g., Bax) via downregulating the Janus kinase/signal transducer and activator of transcription3 (JAK2/STAT3) signaling pathway [55].
Treatment with prodelphinidin B-2 3 -O-gallate, a proanthocyanidin gallate, resulted in the upregulation of key transcription factors such as the soluble Fas ligand (sFasL) and membrane-bound Fas ligand (mFasL), which are responsible for the anti-proliferative and apoptotic activities in NSCLC cells [56,57]. Cinnamtannin D1, an A-type procyanidin trimer, from Rhododendron formosanum extracts has been found to exhibit autophagic effects on NSCLC cells via inhibition of cellular signaling pathways (e.g., mTOR) [58].
These results suggest that plant-derived natural PCs may play a significant role as anti-NSCLC agents by suppressing proliferation, migration, invasion, viability, metastasis, angiogenesis, and promoting apoptosis/autophagy via inhibition/activation of transcription factors and/or multiple cellular signaling pathways induced by α7nAChR in NSCLC cells. Table 1 highlights the molecular mechanisms of PCs in nicotine-induced NSCLC treatment.

Anthocyanins in Nicotine-Induced NSCLC Treatment
ACNs-rich extracts from plant-based foods/medicinal plants at various nontoxic concentrations have exerted therapeutic effects against nicotine-induced NSCLC, as demonstrated by several studies. Delphinidin and cyanidin have been shown to inhibit plateletderived growth factor (PDGF)(AB)-induced VEGF expression in vascular smooth muscle cells through attenuation of the p38 MAPK and c-JUN NH2-terminal kinase (JNK) signaling pathways [59], known as α7nAChR-mediated cascades involved in NSCLC.ACNs extracted from Vitis coignetiae Pulliat inhibit the expression of several transcription and growth factors (e.g., MMP2/9 and VEGF) involved in proliferation, angiogenesis, invasion, and migration of NSCLC cells [60]. The extracts from Morus alba L. (cyanidin 3-rutinoside and cyanidin 3-glucoside) inhibit the migratory and invasive activities of NSCLC cells [61]. Cyanidin-3-glucoside not only inhibits the proliferation, invasion, and migration, but also induces apoptosis. The mechanism underlying such an effect is associated with inhibiting p53-induced gene 3 (TP53I3) expression in NSCLC cells and the downregulation of cellular signaling pathways (PI3K/Akt/mTOR) involved in NSCLC progression [62].
A combination of five ACN extracts (i.e., delphinidin, peonidin, petunidin, cyanidin, and malvidin) from bilberry and blueberry resulted in inhibited growth and metastasis, and induced apoptosis and cell cycle arrest of NSCLC cells in vitro. The mechanism of action of ACNs involves inhibiting activation of multiple signaling pathways, including TNFα-induced NFk B, Notch, Wnt/β-catenin, and their key transcription factors (i.e., cyclin D1/B1, VEGF, p-ERK, bcl-2, and MMP9). Furthermore, when evaluated with an in vivo model using the in vivo xenograft assay, delphinidin alone, and the ACN mixture, resulted in significantly inhibited growth of H1299 cells [65]. ACNs derived from Syzygium cumini L. (known as Indian blackberry) showed significant anti-proliferative effects on NSCLC cells, but the mechanisms for this action have not been observed [66].
These results suggest that ACNs may have a significant role in anti-proliferative, anti-invasive, anti-angiogenic, anti-metastatic, and apoptotic/autophagic effects in NSCLC cells by suppressing the activation of key transcription/growth factors and α7nAChRmediatedcellular signaling pathways. The molecular mechanisms of ACNs in nicotineinduced NSCLC treatment are summarized in Table 2.  In vitro A549 Syzygium cumini L.

Combination of Proanthocyanidins and Anthocyanins with Chemotherapeutics/Radiotherapy in Nicotine-Induced NSCLC Treatment
Several studies suggest that PCs and ACNs used in combination with radiotherapy and/or chemotherapy have the ability to modulate the activity of cellular signaling pathways involved in proliferation, inflammation as well as in anti-apoptosis and/or cell cycle arrest in NSCLC cells. Treatment with PCs extracted from Cinnamomi Cortex inhibits Nrf2-mediated proliferation through promoting sensitivity of NSCLC cells to the cytotoxic action of chemotherapeutic drug etoposide and doxorubicin [67]. A recent study using the CCK8 and ELISA assays reported anti-inflammatory and apoptosis-inducing effects of PCs on NSCLC cells when used in combination with ionizing radiation. The results indicated that such combinations inhibit tumor growth in vivo and reduce interferons/interleukins production and increase pro-apoptotic p53 target genes in vitro via upregulating the MAPK signaling pathway [68]. Delphinidin treatment with γ-ionizing radiation (IR) induced autophagy and apoptosis in NSCLC cells by increasing pro-apoptotic expression (e.g., caspase-3 and LC3), and reduced p-ERK1/2 expression via activation of the MAPK signaling pathway [69]. Cyanidin-3-glucoside, but not pelargonidin-3-glucoside, has been recently reported to inhibit the mRNA level of claudin-2 (CLDN2)-induced chemotherapy resistance to doxorubicin through the downregulation of p-Akt and the upregulation of p38 MAPK, leading to apoptosis and necrotic cell death in NSCLC cells [70]. Table 3 summarizes the molecular mechanisms of PCs and ACNs in combination with radiotherapy/chemotherapy in nicotine-induced NSCLC treatment.

PCs and ACNs as Positive Modulators of Human α7nAChR
Findings of this review suggest that PCs and ACNs exert anti-tumor functions against NSCLC, due to their potential as positive modulators of α7nAChR. Treatment of NSCLC cells with varying concentrations suppresses the growth of these cells by 50% relative to the untreated control (GI 50 ). The concentrations used both in vivo and in vitro studies are not associated with gross/visible toxicity. ACNs overdose possesses no significant cytotoxic effects on NSCLC cells in vivo. The cytotoxicities of anti-NSCLC drugs and/or ionizing radiation are significantly improved in the presence of 100-300 µg/mL Cinnamomi Cortex PCs in vitro, 20 µg/mL grape seed PCs in vivo/vitro, and ≤50 µM ACNs in vitro.
PCs vary in the number, configuration, arrangement and substitution of OH groups on the B ring. In general, all PCs have OH groups on C-3, C-5, and C-7 positions, respectively, and on their B rings [22]. The anti-NSCLC activity of PCs may rely on the number of OH groups on their B-ring, and the OH group at C-3 plays a significant role in their significant anti-tumor ability [71]. Procyanidins have five OH groups on positions C3,5,7,4 ,5 , with one OH group on the C ring and two OH groups on the A and B rings [22]. Procyanidins may interact with α7nAChR binding site and thus could be considered as a potential target for the treatment of nicotine-induced NSCLC.
The number and position of OH and methoxyl groups on the flavylium B-ring of ACNs may play a significant role in the suppression of α7nAChR and its downstream signaling pathways. The presence of one OH group or methoxy substituents on the B-ring may result in a considerable loss of inhibitory effects in NSCLC cells. Delphinidin, which has three OH moieties at C-3 ,4 ,5 of the B-ring, exerts greater inhibitory activity than cyanidin and pelargonidin, which have two and one OH group, respectively. The presence of two OH groups, one methoxyl group in petunidin, and one OH group with methoxy substituents in malvidin and peonidin make them less inhibitory effects on NSCLC cells [25]. Thus, ACNs would be expected to have high levels of effective anti-NSCLC agents in binding to the α7nAChR-mediated signaling pathways, which may result in suppression of the receptor in a concentration-dependent manner.
Understanding conditions associated with nicotine-α7-nAChR signaling might provide an opportunity to explore the therapeutic potential of PCs and ACNs in nicotineinduced NSCLC. Among several types of α subunits, the homomeric α7nAChR has been shown to be the key receptor activating NNK and/or nicotine-mediated cell proliferation, angiogenesis and anti-apoptosis in NSCLC cells in vivo and in vitro [10,72]. High levels of the α7 mRNA levels in squamous cell carcinoma tumors are found in smokers than nonsmokers [73]. NNK has been shown to be a high affinity agonist for α7nAChR in LC cells [74]. Nicotine is converted to NNK via activation of the α7nAChR and βadrenergic receptor (β-AR) [8]. NNK binds to α7nAChR and β-AR causes activation of NSCLC-promoting signaling pathways [10,72]. NNK induces production of ROS in vitro, which causes oxidative DNA damage in NSCLC cells by activating the Wingless-type protein (Wnt) signaling pathway [75]. NNK or nicotine induces NSCLC cell proliferation, invasion and angiogenesis in vivo and in vitro through α7nAChR with activation of COX-2 expression, leading to an upregulation of PGE 2 , which is implicated in NSCLC development [76,77]. NNK enhances proliferation of NSCLC cells in vitro by activating PCNA expression via PI3K/AKT/Stat3/ERK1/2 signaling pathway activation [78]. In vitro, treatment with nicotine causes an induction effect on α7nAChR, along with significant stimulation of p-ERK1/2 cascade, accompanied by promotes the nAChRmediated production of neurotransmitter noradrenaline in the brain, which enhances β-AR signaling-mediated DNA synthesis of NSCLC cells, resulting in promoted cell proliferation [79]. Nicotine influences NSCLC biology involve inhibition of apoptosis induced by chemotherapy and radiotherapy, and induction of proliferation and angiogenesis of NSCLC cells. Such effect is mediated by α7nAChR, which stimulates signaling pathways such as PI3K/Akt/mTOR, suppresses of the mitochondrial death pathway or induces of pro-angiogenic factors [80]. The binding of nicotine to the α7nAChRin vivo/vitro upregulates multiple downstream pathways (e.g., MAPK/ERK, Src, JAK, and STAT) and induces the secretion of growth/angiogenic factors (e.g., EGF and VEGF) in NSCLC cells, and such effect could be mediated by inactivation of p53 expression [9]. Binding of nicotine to α7nAChR enhanced voltage-gated Ca 2+ channels, which result in induction of NSCLC cell angiogenesis by activating of Ca 2+ influx [10,72]. Nicotine induces NSCLC angiogenesis, migration and invasion in vitro by inhibiting HIF-1α and its downstream target gene VEGF. Such effect is mediated via α7nAChR-mediated Src, Ca+2, PI3K, MAPK/ERK1/2, and mTOR signaling pathways activation [81]. Nicotine promotes NSCLC cell proliferation in vitro via protein arrestin, β1 (ARRB1)-mediated activation of E2F transcription factors 1 (E2F1), and Src and Rb-Raf-1 signaling downstream of α7nAChR [82,83]. Nicotine or NNK induces NSCLC cell proliferation in vivo and in vitro by activating Akt phosphorylation and its related downstream substrates, including GSK-3β, mTOR and NFκB [84]. In vitro, proliferation, invasion and angiogenesis of NSCLC cells are found to be promoted by nicotine via α7nAChR with activation of Signal transducer and activator of transcription1 (STAT1), E2F1, as well as induction of Src, PI3K, and Rb-Raf signaling pathways [85]. The apoptotic family members, including Bcl-XL, c-Myc, and Bcl2, are induced in response to nicotine/NNK treatment in NSCLC cells in vitro via α7nAChR-mediated ERK1/2, Raf1 and MAPKs signaling pathways activation, resulting in suppression of apoptosis [86]. In vivo, nicotine or NNK-induced activation of the PI3K/Akt/mTOR pathway via stimulation of α7nAChR inhibits Bax expression [87]. NNK treatment inhibits miRNAs expression in NSCLC cells in vivo. This is observed through upregulation of cytochrome P450 (CYP) 2A3 expression as a mechanism of such effect [88]. NNK induces NSCLC progression in vitro by altering the expression of mismatch DNA repair (MMR), leading to miRNAs deregulation, particularly miR-422a, miR-155, and miR-21 [89]. NNK induces proliferation and inhibits apoptosis in NSCLC cells in vivo by downregulating miR-124 expression, leading to the activation of Akt, thereby enhancing NSCLC progression [90].
PCs may have the ability to inhibit β-amyloid (Aβ) aggregation [91], which is the key hallmark of NSCLC [92,93]. Aβ is one of the key mechanisms responsible for LC, as shown in increased Aβ isoforms in A549 cells, particularly Aβ40 and Aβ42. Inactivation of p53 was found to increase Aβ40 and Aβ42 levels in A549 cells via upregulation of the PI3K/Akt/NFκB signaling pathway and MMP2/9 expression, known to increase viability and reduce apoptosis in NSCLC cells, suggesting that activation of p53 may be helpful in reducing the levels of Aβ40 and Aβ42 responsible for viability and antiapoptosis in NSCLC cells [93]. Aβ40 and Aβ42 contain amino acids serine (Ser) and tyrosine (Tyr) (H 6 DSGY 10 and G 25 SNKG 29 ), which are known to be the major toxic Aβ forms. Ser26 and Tyr10 of Aβ peptides have been shown to interact with acetylcholinesterase (AChE), a cholinergic enzyme-mediated neurotransmitter acetylcholine (Ach) associated with amyloid fibril formation in A549 cells, leading to increased cytotoxic response in these cells [94]. Ach produced by AchE from acetyl-coenzyme A (acetyl-CoA) and choline plays a keyrole as paracrine/autocrine growth factor in the lung homeostasis, which binds to nAChRs on the NSCLC cells [95,96]. The expression of AchE has been reported to be reduced in NSCLC cells. The decrease in AchE activity leads to elevate Ach levels in NSCLC cells, which could promote cell proliferation, migration, invasion, angiogenesis, and reduce apoptosis/autophagy through activating Ca 2+ channels, TGF-β-induced EMT and α7nAChR-mediated signaling pathways such as PI3K/Akt, ERK/MAPK, GSK-3β, and STAT3 [97]. This suggests that Ach may have therapeutic potential in nicotine-induced NSCLC cells by upregulating expression of p53, which promotes apoptosis/autophagy and reduce proliferation and angiogenesis in NSCLC cells. Given that PCs reduce cellular growth and proliferation in NSCLC cells via upregulating p53 expression, targeting such natural compound in treatment regimes might help in downregulating cellular signaling pathways, as the mechanisms involved in Aβ-induced proliferation and anti-apoptosis in NSCLC cells.
Activation of Ach was also found to promote inflammation and pro-inflammatory cytokines in pulmonary tissue [98]. Increased concentrations of pro-inflammatory cytokines (i.e., IL-6, IL-8, IL-1β, and TNF-α) have been reported in NSCLC cells, particularly H460 and A549 cells [99]. The potential mechanism underlying such increases involves activating the α7nAChR-mediated JAK1-dependent STAT3 signaling by receptor tyrosine kinase (RTK) such as VEGFR and mutant-EGFR [100,101]. Therefore, ACNs may have significant anti-inflammatory functions via reducing of Aβ aggregation in NSCLC cells, leading to the modulation of α7nAChR and its downstream signaling pathways.
Previous studies showed that microRNAs (miRs), including miR-205-3p, a class of noncoding RNA molecules, act as potential markers for the progression of NSCLC [102,103]. A recent in vitro experiment has suggested that the Aβ precursor protein-binding family B member 2 (APBB2)-mediated activation of dysregulated miR-205-3p expression in different NSCLC cells, including H460, H1299, 95-D, and A549, resulted in activation of proliferation and inhibition of apoptosis in NSCLC cells [104]. PCs and ACNs may directly bind to Aβ to reduce its aggregation, suggesting that these compounds could be useful in reducing miR-205-3p expression involved in the pathogenesis of NSCLC. PCs and ACNs may thus be useful for their anti-Aβ aggregation activity via reducing miR-205-3p expression, resulting in an inhibition of proliferation and induction of apoptosis in NSCLC cells.
ACNs may bind to the active site of α-L-rhamnosidases (α-R) enzyme, having an inhibitory effect on NSCLC cells by inhibiting the cellular signaling pathways involved in proliferation and anti-apoptosis/autophagy. α-R is involved in ACNs metabolism, which hydrolysis of glycosidic bonds, and thus, may improve the bioavailability as well as the anti-tumor activity of ACNs in nicotine-induced NSCLC cells. For example, α-R enzyme has been demonstrated to catalyze the hydrolysis of cyanidin-3-rutinoside to cyanidin-3-glucoside in black raspberry from Aspergillus spp. [105] and in red mulberry (Morus alba L.) fruit [106], which are known for their anti-NSCLC activity. Thus, α-R may provide highly biologically active ACNs, which suppress proliferation and induction of apoptosis/autophagy in NSCLC cells.
Taken together, nicotine-mediated activation of cell proliferation, migration, invasion, angiogenesis, and inhibition of apoptosis in NSCLC cells can be modulated when PCs and ACNs interact with the active site of α7AChR, displaying complex modulation of several transcription/growth factors via signaling pathways involved in α7nAChR activation, suggesting that such compounds may have a significant role in the treatment of NSCLC ( Figure 2 Previous studies showed that microRNAs (miRs), including miR-205-3p, a class of noncoding RNA molecules, act as potential markers for the progression of NSCLC [102,103]. A recent in vitro experiment has suggested that the Aβ precursor protein-binding family B member 2 (APBB2)-mediated activation of dysregulated miR-205-3p expression in different NSCLC cells, including H460, H1299, 95-D, and A549, resulted in activation of proliferation and inhibition of apoptosis in NSCLC cells [104]. PCs and ACNs may directly bind to Aβ to reduce its aggregation, suggesting that these compounds could be useful in reducing miR-205-3p expression involved in the pathogenesis of NSCLC. PCs and ACNs may thus be useful for their anti-Aβ aggregation activity via reducing miR-205-3p expression, resulting in an inhibition of proliferation and induction of apoptosis in NSCLC cells.
ACNs may bind to the active site of α-L-rhamnosidases (α-R) enzyme, having an inhibitory effect on NSCLC cells by inhibiting the cellular signaling pathways involved in proliferation and anti-apoptosis/autophagy. α-R is involved in ACNs metabolism, which hydrolysis of glycosidic bonds, and thus, may improve the bioavailability as well as the anti-tumor activity of ACNs in nicotine-induced NSCLC cells. For example, α-R enzyme has been demonstrated to catalyze the hydrolysis of cyanidin-3-rutinoside to cyanidin-3-glucoside in black raspberry from Aspergillus spp. [105] and in red mulberry (Morus alba L.) fruit [106], which are known for their anti-NSCLC activity. Thus, α-R may provide highly biologically active ACNs, which suppress proliferation and induction of apoptosis/autophagy in NSCLC cells.
Taken together, nicotine-mediated activation of cell proliferation, migration, invasion, angiogenesis, and inhibition of apoptosis in NSCLC cells can be modulated when PCs and ACNs interact with the active site of α7AChR, displaying complex modulation of several transcription/growth factors via signaling pathways involved in α7nAChR activation, suggesting that such compounds may have a significant role in the treatment of NSCLC ( Figure 2).

Limitations
Although the available in vivo and in vitro studies suggest a therapeutic potential of PCs and ACNs in nicotine-induced NSCLC, these studies are used different dosage regimens in the treatment, which questions the reliability of results from these studies. Different doses of PCs and ACNs may affect diverse transcription factors and multiple cellular signaling pathways, and thereby affecting targeted nicotine-induced NSCLC therapy. The optimal effective dose of PCs and ACNs are difficult to determine, as it varied between studies. PCs and ACNs could be effective in nicotine-induced NSCLC treatment if used at concentration of up to 100 µM/µg/mL, but further studies are needed to apply this in vitro anti-NSCLC effects.

Conclusions
PCs and ACNs are flavonoid pigments with anti-NSCLC effects that exist in various plant-based foods and/or medicinal plants. The potential molecular mechanisms by which PCs and ACNs from plants-based foods and medicinal plants exert anti-tumor activity with therapeutic potential in nicotine-induced NSCLC have not been elucidated. Treatment with PCs and ACNs at various nontoxic concentrations have demonstrated several activities against nicotine-induced NSCLC, including anti-proliferative, anti-migration, antimetastatic, anti-invasive, anti-angiogenic, and apoptosis/autophagy induction. PC-rich extracts from grape seed and/or Cinnamomi Cortex used in combination with irradiation/chemotherapy may have useful anti-proliferative, anti-inflammatory and apoptotic efficacy against nicotine-induced NSCLC. Delphinidin and cyanidin may promote apoptotic/autophagic activity by enhancing the chemosensitivity and/or radiosensitivity of NSCLC cells.
Nicotine and/or NNK may contribute to NSCLC development by promoting cell proliferation, migration, invasion, angiogenesis, and inhibiting apoptosis. Such effects may occur through α7nAChR-mediated signaling pathways activation. PCs and ACNs may act as selective agonists of α7nAChR, where they can inhibit the activation of α7nAChR in NSCLC cells. PCs and ACNs may directly bind to α7nAChR, suggesting that these compounds could be useful in modulating multiple transcription/growth factors and signaling pathways. PCs may act as anti-NSCLC agents through inhibiting excessive accumulation of Aβ involved in nicotine-induced NSCLC. In addition, the α-R enzyme involved in deglycosylation of ACNs in plant-based foods may play a key therapeutic role in nicotine-induced NSCLC, as they are involved in the anti-proliferative and apoptotic/autophagic activities against NSCLC cells. Therefore, plant-based foods and/or medicinal plants-derived PCs and ACNs may have therapeutic efficacy as anti-NSCLC medicinal agents in smokers due to their anti-proliferative, anti-angiogenic, anti-migration, anti-invasive, anti-inflammation, anti-metastatic, and apoptosis/autophagy activities, but further studies are needed to better understand the underlying mechanisms.


Transport of Prions in the Peripheral Nervous System: Pathways, Cell Types, and Mechanisms

Prion diseases are transmissible protein misfolding disorders that occur in animals and humans where the endogenous prion protein, PrPC, undergoes a conformational change into self-templating aggregates termed PrPSc. Formation of PrPSc in the central nervous system (CNS) leads to gliosis, spongiosis, and cellular dysfunction that ultimately results in the death of the host. The spread of prions from peripheral inoculation sites to CNS structures occurs through neuroanatomical networks. While it has been established that endogenous PrPC is necessary for prion formation, and that the rate of prion spread is consistent with slow axonal transport, the mechanistic details of PrPSc transport remain elusive. Current research endeavors are primarily focused on the cellular mechanisms of prion transport associated with axons. This includes elucidating specific cell types involved, subcellular machinery, and potential cofactors present during this process.

Introduction
Prion diseases are protein misfolding disorders that can have an infectious, sporadic, or genetic etiology, and lead to the slow, progressive, and inevitable demise of the host through neuronal degeneration of the CNS [1][2][3]. Although prion diseases were initially identified early in the 20th century, the exact nature of the infectious agent responsible for such disorders has only recently been determined. Initially described as slow viruses, prion diseases were thought to be caused by a slowly progressive viral infection [4][5][6][7]. It is now established that prion diseases are caused by a proteinaceous infectious particle, or prion [1,8,9]. Importantly, prions do not contain a nucleic acid genome like viral particles, but have templating abilities that allow propagation in the host [10,11].
The prion protein (PrP C ) is an endogenous protein coded by the PRNP gene that is present within most mammalian species with paralogues in certain reptiles such as turtles [12]. Although the exact functions of PrP C are unknown, there is evidence that it participates in a variety of roles including neurogenesis, neuronal development, and synaptic function [13][14][15][16]. In the infectious prion disease etiology, PrP C undergoes an aberrant conformational change into misfolded prion protein (PrP Sc ) that is induced by the interaction of exogenous PrP Sc with endogenous PrP C . In sporadic prion diseases PrP C is thought to spontaneously adopt the self-propagating infectious PrP Sc conformation. In familial forms of prion diseases, mutations in the PRNP gene result in an increased propensity of PrP C adopting the PrP Sc conformation compared to wild type PrP C [17,18]. The tertiary and/or quaternary structure of PrP Sc results in an increased resistance to degradation by proteases, heat, pH, and various other environmental factors compared to PrP C [19][20][21][22][23][24][25]. In addition, whereas PrP C is considered a monomer, PrP Sc can aggregate within cells leading to cellular dysfunction, neurodegeneration, gliosis, and spongiosis [2,3,17,26,27]. This CNS pathology is what ultimately leads to the clinical signs of disease (ataxia, tremor, behavioral changes, lethargy, etc.) and the death of the host.
The structural conformation of PrP Sc is hypothesized to encode prion strain diversity [28]. Prion strains are operationally defined as heritable strain-specific phenotypes of disease that are characterized by incubation period, clinical signs of disease, CNS pathology, and distribution of prions in the host [29,30]. Prion strain-specific biochemical and biological properties of PrP Sc can include electrophoretic mobility, stability of PrP Sc in the presence of protein denaturants, PrP Sc aggregate size distribution, and rate of PrP Sc formation [30][31][32][33]. It is unclear how these differences in strain-specific properties of PrP Sc result in differences in the phenotype of disease. Recent work, however, suggests that PrP Sc particle size can influence prion formation efficiency and PrP Sc clearance and that strain-specific ratios of these PrP Sc particle sizes may provide a mechanistic basis for strain-specific rates of prion formation, incubation period of disease, and tissue tropism [34][35][36].
Prion diseases affect a wide variety of mammalian species. Notable examples of prion diseases with an infectious etiology include kuru, variant Creutzfeldt Jacob disease (vCJD), bovine spongiform encephalopathy (BSE), transmissible mink encephalopathy (TME), chronic wasting disease (CWD), and scrapie. Kuru occurs in the Fore people of Papa New Guinea and is thought to be caused by the consumption of nervous tissues from individuals afflicted with CJD via a practice of mortuary feasts of deceased family members [27,[37][38][39][40][41][42][43][44][45]. BSE was first described in the United Kingdom in 1987 [46] and was propagated in cattle by the feeding of BSE-tainted meat and bone meal back to cattle [47,48]. The BSE epidemic caused a public health crisis in Europe, resulting in the transmission of BSE to humans through the emergence of vCJD [49][50][51]. TME is a prion disease of ranch-raised mink that was initially thought to be caused by feeding mink scrapie-infected sheep tissue [52][53][54]. Epidemiological and experimental evidence from the Stetsonville outbreak of TME, however, suggested that TME is caused by feeding mink downer cattle infected with an unrecognized BSE-like disease [55,56]. Subsequent studies supporting this hypothesis showed that atypical sporadic L-type BSE is the source of TME [57]. CWD is an emerging prion disease of cervids that was first described in a captive deer facility in Colorado in 1969 and was officially characterized as a prion disease in 1980 [58,59]. CWD has continued to spread throughout the United States and North America and is currently found in 27 states and 3 Canadian provinces [60,61]. Recently, CWD cases have been identified in Norway, Sweden, and Finland. Compared to North American CWD, the Scandinavian CWD cases have PrP Sc and transmission properties suggesting that it is a unique strain of CWD [24,[62][63][64][65][66][67]. Notably, CWD prions persist in the environment and are highly contagious resulting in horizontal transmission in both captive and free-ranging cervid populations [24,25,68]. Scrapie in sheep and goats was described as early as the 18th century and is still present in sheep and goat populations today [7,37,[69][70][71][72][73][74][75][76]. Regarded as the prototypical prion disease, scrapie has played a large role in understanding prion disease pathogenesis. Like CWD, it can persist in the environment and spread through horizontal transmission [77][78][79]. Notably, placentas of scrapie positive ewes contain prion infectivity and may be a source of infection in utero (vertical transmission) or through environmental contamination early in the lambing stages [80].

Intraocular Inoculation of Prions
Prions spread along defined neuroanatomical pathways following the intraocular route of infection [81,82]. Evidence that the prion agent is transported in CNS tissues via known neural pathways was established by determining the temporal and spatial spread of prion replication and pathology following intraocular injection of mice with the scrapie agent. Inoculation of the retina resulted in detection of prion infectivity in the contralateral superior colliculus (SC) via the optic nerve ( Figure 1, [81,83]). In addition to the contralateral SC, scrapie infectivity was also identified in the contralateral lateral geniculate nucleus (LGN) and the contralateral visual cortex at later time points post-  (Figure 1, [82,84]). Importantly, infectivity and vacuolation pathology was greater in the contralateral SC and LGN compared to the ipsilateral SC and LGN [82,85]. Since the rodent visual pathways almost completely decussate [86][87][88], this suggested the transport of prions from the injection site in the eye to the CNS occurs along defined neuroanatomical tracks. To support this hypothesis, enucleation of the retina just prior to or immediately after scrapie inoculation delayed the onset of clinical signs of disease, the increase in infectivity, and the development of vacuolation pathology [85]. This observation indicates that when the retina was removed, scrapie prions were not able to effectively access CNS structures as the neuronal pathway was eliminated. Following this centripetal (periphery to CNS) prion spread, a centrifugal (CNS to periphery) prion spread was also observed. teral SC, scrapie infectivity was also identified in the contralateral lateral geniculate nucleus (LGN) and the contralateral visual cortex at later time points post-infection ( Figure  1, [82,84]). Importantly, infectivity and vacuolation pathology was greater in the contralateral SC and LGN compared to the ipsilateral SC and LGN [82,85]. Since the rodent visual pathways almost completely decussate [86][87][88], this suggested the transport of prions from the injection site in the eye to the CNS occurs along defined neuroanatomical tracks.
To support this hypothesis, enucleation of the retina just prior to or immediately after scrapie inoculation delayed the onset of clinical signs of disease, the increase in infectivity, and the development of vacuolation pathology [85]. This observation indicates that when the retina was removed, scrapie prions were not able to effectively access CNS structures as the neuronal pathway was eliminated. Following this centripetal (periphery to CNS) prion spread, a centrifugal (CNS to periphery) prion spread was also observed. At later timepoints post infection, the uninoculated contralateral optic nerve and retina developed scrapie infectivity [81] indicating prions spread from the peripheral structures to central structures, and then spread back into unaffected peripheral structures ( Figure 1). Since this phenomenon is observed later in the disease process it may be less efficient compared to the initial spread of infectivity. As the major neuronal connections of the inoculated retina are to the contralateral SC and LGN [82,86], scrapie prions will more easily spread along these pathways and lead to earlier and more significant pathology. In contrast, the projection to the ipsilateral SC and LGN is more modest from the inoculated retina, and there may also be minor connections between the bilateral SC and LGN structures allowing for attenuated prion spread between the structures [86,[89][90][91]. This results in prion invasion and the spread of pathology that takes longer (via more minor neuronal connections) and is less prominent at the end stage of disease. Once infected, the ipsilateral SC and LGN can transport prion infectivity to the uninoculated retina and its respective optic nerve via retrograde transport or the ipsilateral visual cortex . As the animal approaches clinical endpoint, the ipsilateral SC, LGN, and visual cortex are also affected to a lesser extent (blue structures). This is thought to be due to reciprocal connections between the contralateral SC (solid black arrow; 3) and LGN (dashed black arrow; 3) and ipsilateral anterograde spread from the inoculated retina (dashed red line; 4). The uninoculated optic nerve and retina are also affected through retrograde prion spread (solid blue line; 5). Image created with BioRender.com, accessed on 28 February 2022.
At later timepoints post infection, the uninoculated contralateral optic nerve and retina developed scrapie infectivity [81] indicating prions spread from the peripheral structures to central structures, and then spread back into unaffected peripheral structures ( Figure 1). Since this phenomenon is observed later in the disease process it may be less efficient compared to the initial spread of infectivity. As the major neuronal connections of the inoculated retina are to the contralateral SC and LGN [82,86], scrapie prions will more easily spread along these pathways and lead to earlier and more significant pathology. In contrast, the projection to the ipsilateral SC and LGN is more modest from the inoculated retina, and there may also be minor connections between the bilateral SC and LGN structures allowing for attenuated prion spread between the structures [86,[89][90][91]. This results in prion invasion and the spread of pathology that takes longer (via more minor neuronal connections) and is less prominent at the end stage of disease. Once infected, the ipsilateral SC and LGN can transport prion infectivity to the uninoculated retina and its respective optic nerve via retrograde transport or the ipsilateral visual cortex via anterograde transport ( Figure 1). Overall, these data indicate the anterograde transport of scrapie prions through established

Extraneural Inoculation of Prions
Prions spread along defined neuroanatomical pathways following extraneural routes of infection. Intraperitoneal (i.p.) injection of scrapie resulted in scrapie infectivity that was first detected in the spleen [92][93][94]. Within the spleen and other LRS tissues, PrP Sc has been shown to accumulate on follicular dendritic cells (FDCs) and within tingible body macrophages (TBM, [95,96]). Elimination of FDCs results in a failure of prions to establish infection in secondary LRS tissues and suggests that FDCs actively replicate PrP Sc while TBMs scavenge PrP Sc [96][97][98]. Accumulation of PrP Sc on FDCs has been shown to occur for the duration of prion infection and does not result in cellular dysfunction [99][100][101][102]. Evidence suggests that PrP Sc produced by FDCs can infect sympathetic nerves that innervate LRS tissue, as denervation or increasing the physical distance between FDCs and nerve fibers can extend the incubation period of disease or prevent disease transmission [103]. Following i.p. inoculation, prions were found to spread from the spleen to the thoracic spinal cord via the splenic sympathetic axons, with subsequent spread caudally and rostrally to the lumbar and cervical spinal cord, respectively, prior to detection in the brain ( Figure 2A, [83,94,104]). In the brain, scrapie infectivity was first detected in caudal brain structures such as the medulla and the midbrain and eventually spread to the cerebellum and the cerebral cortex.
Per os infection of prion agent results in neuroinvasion along defined anatomical pathways that involve the sympathetic and parasympathetic pathways innervating the gut. Intragastric prion inoculation of mice resulted in detection of infectivity in the Peyer's patches of the gut, followed by spread of infectivity via the myenteric plexus and the enteric nervous system (ENS) to the thoracic spinal cord and finally the brain ( Figure 2B, [105]). Consistent with this pathway of prion transport, splenectomy did not prolong the incubation period following intragastric inoculation. In sheep naturally infected with scrapie, PrP Sc immunoreactivity was first detected in the Peyer's patches. Later in the disease course, PrP Sc immunoreactivity was detected in the myenteric plexus and ENS, the celiac sympathetic ganglia near the thoracic spinal cord, subsequently in the intermediolateral cell column of the thoracic spinal cord, and finally in the brain [106]. Simultaneously, PrP Sc immunoreactivity was also found in the parasympathetic nodose ganglia and subsequently in the dorsal motor nucleus of the vagal nerve (DMNV). Oral BSE challenge of cattle also identified PrP Sc immunoreactivity and infectivity that spread from the ENS to the brain via sympathetic pathways involving the thoracic spinal cord and parasympathetic pathways involving the vagus nerve [107][108][109]. Oral inoculation of hamsters with 263K scrapie prions recapitulated many of the findings from the sheep studies where 263K PrP Sc was detected in the ENS, then in the sympathetic ganglia, thoracic spinal cord, and later in the medulla [110][111][112]. Importantly, scrapie was also observed to invade the CNS through the DMNV independent of the thoracic spinal cord pathway [113,114]. In this case, scrapie prions were hypothesized to invade the ENS, then travel through the vagal nerve parasympathetic pathway, directly invade the DMNV, and subsequently transneuronally spread throughout the CNS ( Figure 2B). Neuroinvasion via the DMNV is also observed in deer per os inoculated with CWD [115,116]. Overall, these data indicate the retrograde spread of PrP Sc through the autonomic nervous system via sympathetic and parasympathetic pathways after extraneural inoculation. fibers can extend the incubation period of disease or prevent disease transmission [103]. Following i.p. inoculation, prions were found to spread from the spleen to the thoracic spinal cord via the splenic sympathetic axons, with subsequent spread caudally and rostrally to the lumbar and cervical spinal cord, respectively, prior to detection in the brain (Figure 2A, [83,94,104]). In the brain, scrapie infectivity was first detected in caudal brain structures such as the medulla and the midbrain and eventually spread to the cerebellum and the cerebral cortex.

Intraneural Inoculation of Prions
Prions spread along defined neuroanatomical pathways following inoculation into peripheral nerves [117,118]. Unilateral inoculation of scrapie prions into the sciatic nerve of hamsters resulted in a significantly shortened incubation time compared to all other inoculation routes with the exception of intravenous and intracerebral routes [117]. Importantly, scrapie infectivity was detected in spinal cord segments at earlier timepoints post-infection compared to other extraneural infection (intraperitoneal, oral, subcutaneous, etc.) routes suggesting that scrapie prions were able to bypass the spleen and other LRS tissues, and directly invade and spread via axonal networks ( Figure 2C). Consistent with these studies, a detailed analysis of the temporal and spatial spread of hyper (HY) PrP Sc following inoculation of the sciatic nerve revealed that prions initially spread to ipsilateral ventral motor neurons in the lumbar spinal cord followed by transport along the rubrospinal,  [118]. Axons of these four motor pathways synapse either directly or indirectly on VMNs [119]. HY PrP Sc was detected in progressively more rostral spinal cord segments starting with the T10-T13 segment corresponding to where the sciatic nerve enters the lumbar spinal cord ( Figure 2C). Within the brain, PrP Sc was detected in the ventral portion of the contralateral RN (origin of the rubrospinal tract) which decussates and has direct projections to ventral motor neurons found in the lumbar spinal cord associated with the inoculated sciatic nerve. Other regions that are associated with the sciatic nerve motor pathways were also affected including the contralateral hindlimb motor cortex (origin of the corticospinal tract) and the ipsilateral lateral vestibular nucleus (origin of the vestibulospinal tract). Since HY PrP Sc was not detected in the spleen following sciatic nerve inoculation, neuroinvasion and subsequent spread was via the sciatic nerve and not from sympathetic innervation from the spleen [118,120]. In addition to HY, sciatic nerve inoculation experiments with drowsy (DY) or 139H strain of prions, resulted in transport of PrP Sc along the same four descending motor pathways [119,121]. Intralingual inoculation of HY has demonstrated PrP Sc deposition in the hypoglossal nucleus, consistent with PrP Sc spread via the hypoglossal nerve [122]. Overall, PrP Sc is transneuronally transported along defined neuroanatomical pathways in the anterograde (intraocular inoculation) or retrograde (intraperitoneal, intragastric, or intraneural) direction. This is also independent of prion strain, suggesting this is a common feature of prions. A summary of the discussed transport pathways can be found in Table 1.

Cell Types Involved in Prion Transport
While it is well-established that prions invade and spread through the peripheral nerves to access the CNS, the mechanism by which this phenomenon occurs has remained elusive. Neurotropic viruses such as rabies and pseudorabies travel from the periphery to the CNS via axons. For example, the rabies virus utilizes microtubule polymerization as it undergoes retrograde transport along motor neuron axons [123]. Herpes viruses also utilize microtubules to spread along both sensory and motor neurons [124]. Since neurotropic viruses and prions spread along established neuroanatomical pathways after infection, the processes utilized by these viral agents serve as a starting point to elucidate the mechanisms involved in prion transport.
The exact cellular location of PrP Sc within neuronal pathways is unknown. In the nervous system, PrP C is expressed not only in neurons and axons, but it has also been found in Schwann cells that myelinate axons in the PNS, oligodendrocytes that myelinate axons in the CNS, as well as astrocytes and microglia that support CNS neurons [16,125]. PrP Sc was localized to the adaxonal membrane in peripheral nerves of scrapie-infected sheep [126] and was also described in adaxonal location in peripheral nerves of CJD patients after immunohistochemical analyses [127], implicating neuronal axons as a pathway of PrP Sc spread. In addition, PrP Sc aggregates traveled along neuritic projections in in vitro neuronal cultures [128]. To complement this finding, Schwann cells with ablated PrP C expression using two different transgenetic mouse models did not slow the progression and incubation period of scrapie after intraperitoneal inoculation in mice [129,130]. This further strengthens the overarching finding that prions invade the CNS through peripheral neuronal pathways and utilize axons to propagate along these pathways towards central structures. These studies also suggest that the contribution of Schwann cells and oligodendrocytes are not required for PrP Sc transport. There is still speculation on the exact location of PrP Sc within an axonal segment. PrP C has been noted as a GPI anchored membrane protein [13], but its conversion into PrP Sc with altered properties may translocate it to another area on the axon, and confocal laser scanning imaging has detected PrP Sc on the axonal periphery and within the axonal cytoplasm [131]. PrP Sc populations have also been localized to endosomal and lysosomal compartments ( Figure 3A), suggesting that PrP Sc may also utilize intracellular membranous vesicles for movement within cells [128,132]. Interestingly, it was found that only a small fraction of PrP Sc was associated with membrane-bound vesicles, indicating free PrP Sc is also present within the cell. These findings suggest different roles of PrP Sc based on axonal location and cell compartment where one population of PrP Sc is primarily transported to other structures while another population aggregates and causes disease pathology. Alternatively, this may indicate multiple modalities of PrP Sc transport that utilize different subcellular mechanisms.  include retrograde PrP Sc transport on dynein motors (A) in a traditional axonal transport paradigm. Of note, PrP Sc may be free aggregates or contained within membranous vesicles such as endosomes or lysosomes. A "domino-like" mechanism has also been proposed (B), where PrP Sc initiates the conversion of neighboring PrP C , and this conversion chain continues down the length of the axonal projection. Image created with BioRender.com, accessed on 28 February 2022.

Prion Transport Rate and Machinery
The velocity of PrP Sc transport and whether prions utilize a fast or slow mode of axonal transport may provide insight into the mechanisms of prion transport. Axonal transport includes two molecular motors, kinesin and dynein. Kinesin is the main component involved in anterograde transport (from soma to synapse) while dynein takes part in retrograde transport (from synapse to soma). Axonal transport can also be divided into two main types, fast axonal transport and slow axonal transport which includes two components, a and b. Both transport mechanisms utilize kinesin and dynein motors and are differentiated based on their speeds, cargoes, subtypes of kinesin and dynein, and cofactors [133]. Fast axonal transport moves organelles and vesicles containing neurotransmitters at upwards of 400 mm/day. Slow axonal transport primarily moves cytoskeletal elements such as microtubules and neurofilaments (slow component a) or cytosolic proteins (slow component b) at less than 8 mm/day [134].
Estimates of the rate of prion spread is consistent with slow axonal transport mechanism ( Figure 3, [104,[117][118][119]). Importantly, prion spread was determined by assaying for prion infectivity via animal bioassay or immunodetection of PrP Sc . These assays do not discriminate between the original inoculum and newly converted prions, therefore, the time to detection of infectivity or PrP Sc may be a combination of both transport and replication, hence the use of the term prion "spread" (Figure 4). Initial estimates of prion spread calculated from the detection of scrapie infectivity in different tissues at various times post infection determined that mouse adapted scrapie prions spread approximately 1-2 mm/day [104,117]. Subsequent studies corroborated that the spread of prions in the nervous system is consistent with slow axonal transport [85,112]. In a series of studies that utilized the sciatic nerve inoculation paradigm and three hamster-adapted rodent prion strains, strain-specific differences in the rate of PrP Sc spread were identified. In these studies, the rate of spread of HY, 139H and DY PrP Sc in the nervous system, as determined by immunohistochemistry, was 1.10 ± 0.11, 1.80 ± 0.27 and 4.14 ± 0.35 mm/day, respectively [119,121]. While these values were noted to be statistically different, they are still within the range of slow axonal transport. The reason for the observed differences is unknown, however these strains were shown to have differing rates of prion formation with the HY strain having a faster rate of prion formation compared to 139H and DY [34]. It is possible, that all three strains move along axons at the same velocity but since HY PrP Sc amplifies faster than PrP Sc from the other two strains, it may reach a level of abundance that is detectable by IHC faster than either DY or 139H (Figure 4). Alternatively, these findings could indicate that different strains utilize different host mechanisms of PrP Sc transport. Overall, the actual velocity of PrP Sc transport is unknown, as PrP Sc may traffic to tissues much earlier in the disease process that can only be elucidated through experiments that directly detect inoculum PrP Sc . A summary of prion transport rate studies can be found in Table 1.
The role of endogenous PrP C in the transport of PrP Sc from the periphery to the CNS is poorly understood. Landmark studies confirmed the requirement of PrP C for successful prion infection [135,136] since PrP C expression is a necessity for prion formation. Studies utilizing PrP C expressing neural grafts found that not only did the target CNS tissue require PrP C for successful infection and pathology, but the peripheral pathways leading to the tissue also required PrP C expression to propagate pathology from the periphery to the CNS [137,138]. This may indicate a need for a continuous supply of PrP C from the site of inoculation to the target site in the CNS for successful infection and clinical pathology of Viruses 2022, 14, 630 9 of 14 CNS structures. It is possible that PrP C is required for axonal transport or that PrP C must be present along the whole tract to serve as a substrate for continual conversion of PrP C to PrP Sc all the way to the CNS in a "domino-like" fashion ( Figure 3B). Alternatively, the failure to establish infection in the PrP C containing grafts following peripheral inoculation may be due to the grafts not being synaptically connected to the peripheral neuronal pathways and therefore not allowing PrP Sc to invade the graft. Interestingly, multiple cell culture studies have concluded that although endogenous PrP C may play a role in initial PrP Sc uptake into the cell, it is not necessary for infection of the cell or PrP Sc transport between cells [139,140]. Evidence from imaging studies conducted on in vitro primary neuronal cultures found that PrP C knockout neuronal cells could still take up and transport PrP Sc within neurites [128] so PrP Sc may not require endogenous PrP c for transport. strains, strain-specific differences in the rate of PrP Sc spread were identified. In these studies, the rate of spread of HY, 139H and DY PrP Sc in the nervous system, as determined by immunohistochemistry, was 1.10 ± 0.11, 1.80 ± 0.27 and 4.14 ± 0.35 mm/day, respectively [119,121]. While these values were noted to be statistically different, they are still within the range of slow axonal transport. The reason for the observed differences is unknown, however these strains were shown to have differing rates of prion formation with the HY strain having a faster rate of prion formation compared to 139H and DY [34]. It is possible, that all three strains move along axons at the same velocity but since HY PrP Sc amplifies faster than PrP Sc from the other two strains, it may reach a level of abundance that is detectable by IHC faster than either DY or 139H (Figure 4). Alternatively, these findings could indicate that different strains utilize different host mechanisms of PrP Sc transport. Overall, the actual velocity of PrP Sc transport is unknown, as PrP Sc may traffic to tissues much earlier in the disease process that can only be elucidated through experiments that directly detect inoculum PrP Sc . A summary of prion transport rate studies can be found in Table 1.  The role of endogenous PrP C in the transport of PrP Sc from the periphery to the CNS is poorly understood. Landmark studies confirmed the requirement of PrP C for successful prion infection [135,136] since PrP C expression is a necessity for prion formation. Studies utilizing PrP C expressing neural grafts found that not only did the target CNS tissue require PrP C for successful infection and pathology, but the peripheral pathways leading to the tissue also required PrP C expression to propagate pathology from the periphery to the CNS [137,138]. This may indicate a need for a continuous supply of PrP C from the site of inoculation to the target site in the CNS for successful infection and clinical pathology of . Prion Transport and Spread. PrP Sc can traffic to its target directly (transport) or can convert endogenous PrP C anywhere along the pathway and amplify (spread). Currently, detection methods such as immunohistochemistry (IHC) or Western blot (WB) can only measure the spread of PrP Sc because threshold levels of PrP Sc are required for successful detection. Moreover, this spread has been demonstrated to vary between prion strains and can influence calculation of transport/spread. More sensitive measurement techniques can approximate transport more accurately (requires lower PrP Sc threshold for detection or even directly measure PrP Sc transport). Note: Light green dots in coronal brain sections represents the original inoculum PrP Sc , while the black dots represent newly replicated PrP Sc . Image created with BioRender.com, accessed on 15 March 2022.

Conclusions
Studies of the peripheral to central transport of prions have yielded many results of prion spread into the CNS. As evidenced by the studies above, prions spread along defined neuronal projections that innervate the inoculation site. Peripheral extraneural inoculation allows many prion strains to replicate in the LRS and travel along sympathetic projections toward the spinal cord where the agent spreads rostrally toward the brain. In the case of per os inoculation, prions spread along sympathetic and parasympathetic projections of the ENS and invade the spinal cord and DMNV, respectively. Peripheral intraneural injections demonstrate prion spread along defined neuroanatomical pathways that connect peripheral axonal pathways with specific brainstem nuclei and/or cortical areas. Currently, efforts are being undertaken to understand mechanisms behind prion spread. So far, PrP C has been demonstrated to be a necessary part of successful prion infection, and evidence has indicated prions utilize axonal networks for peripheral to central transport. The approximate spread of prions is consistent with a slow axonal transport mechanism, yet the exact speed, subcellular mechanisms, and specific cofactors behind this transport phenomenon are still unknown. Overall, future research should focus on elucidating these mechanisms to further our understanding of peripheral prion disease pathogenesis.

Conflicts of Interest:
The authors declare no conflict of interest.